The study of physical and functional characteristics of biological tissues (histology) often involves affixing cells or thin tissue sections to a support such as a glass microscope slide. The affixed tissue materials are then subjected to various procedures such as differential staining to reveal specific features of the tissue upon subsequent microscopic examination.
Many of these procedures, such as staining, can be done by immersing the slide in a large excess of the test solution containing the stain. Total slide immersion is not feasible for some procedures due to the attributes of either the test solution (e.g., cost, scarcity, safety) and/or the procedure (e.g., the need for rapid temperature changes). In such cases, it is desirable to use a minimal volume (e.g., less than 0.1 ml) of the test solution in direct contact with the tissue being investigated. Such minimal volume procedures include: nucleic ambient temperatures. The test solution also may be rapidly cycled among a variety of temperatures ranging up to nearly 100.degree. C. Because the concentration of components in the test solution is critical to success of the procedure, there is an obvious need to control evaporation of the water from the minimal volume test solution on the microscope slide during the procedures.
Various methods have been devised to control evaporation from microscope slides. For some procedures where the temperature is held constant (e.g., nucleic acid hybridizations), the slide with a drop of the test solution over the tissue material can simply be placed in a humidified chamber at the appropriate temperature. This works adequately well but requires a temperature controlled instrument with an appropriate humidification source. If the procedure requires a changing temperature regime, however, humidified chambers are not appropriate due to problems associated with condensation and evaporation occurring during thermal transitions.
An obvious method of eliminating evaporation is to create a small chamber directly on the microscope slide over the tissue material. The chamber contains the test solution, excludes air and is sealed by some mechanism so that evaporation is minimized or eliminated. Traditionally, chambers have been made by positioning a standard cover glass over the tissue material with a layer of the test solution contained between the cover glass and the slide. Microscope slides in common use may have printed surface patterns which define separate areas of the slide and provide a raised frame to hold the cover glass a fixed distance above the surface of the slide. To seal the chamber, some practitioners immerse the slide with the test solution over the tissue and a cover glass in place in a small volume of a non-water miscible fluids, e.g., mineral oil. This method has a number of disadvantages. It does not guarantee that the aqueous test solution will stay over the tissue material. It is difficult to perform rapid thermal cycling. Furthermore, it is very messy and requires extensive rinsing with non-aqueous solvents to remove the mineral oil after the procedure is completed.
To create a sealed chamber without the use of mineral oil in prior art practices, the edges of the cover glass have been sealed to the slide by a variety of means well known in the art including fingernail polish, rubber cement and various commercially available glues. These sealing methods are tedious, messy, can involve the inhalation by the user of organic solvents, are prone to failure, or worse, they can "poison" the test reactions if not applied correctly. In addition, fingernail polish is the only one of these sealing methods suitable for the higher temperature thermal cycling procedures. Another problem with these sealants is experienced when removing them and the cover glasses after the procedure is ended. Removal requires soaking in solvents (e.g., ethanol or xylene) and/or careful manipulation with a razor blade followed by scraping to remove residual sealant from the slide.
Various commercially available chambers for affixing to microscope slides have been marketed as alternatives to the above mentioned sealants. These commercially available chambers use: a) simple adhesion of a smooth rubber gasket to the glass slide to affix the chamber (such as a product called Probe Clips.TM., available from Grace BioLabs); b) a pressure sensitive adhesive around the periphery of a molded plastic funnel shaped device (including a product called Gene Wells.TM., available from Techne, Inc., Princeton, N.J.; and a product called Gene Cone.TM., available from Gene Tec Corp., Durham, N.C.); or c) small silicone chambers "sealed" by pressure to the slide using a stainless steel clip mechanism (a product called Amplicover.TM. Discs and Clips, available from the Applied Biosystems Division of Perkin Elmer Corp., Foster City, Calif.), described in U.S. Pat. No. 5,364,790 issued Nov. 15, 1994 and assigned to the Perkin-Elmer Corporation. The Probe Clips.TM. product has been found to provide insufficient adhesion to maintain a seal at elevated temperatures especially on slides which are not extremely clean and therefore cannot be used for most thermal cycling procedures. The Gene Cones.TM. product has been found to require a heat step of 95.degree. C. for 15 seconds to fix the adhesive before a test solution can be added. This heat step is not only inconvenient but may be incompatible with some tissue materials. In addition, each Gene Cone.TM. product covers a limited area of the slide and the cone extends several millimeters above the slide surface, rendering this product incompatible with a number of commercially available slide thermal cycling instrumentation. The Amplicover.TM. Disks and Clips products are part of an integrated system requiring the use of special thick slides (thus limiting their utility for archival histological preparations), special disposables and a costly assembly tool as well as a specifically designed thermal heating block. In addition, the Amplicover.TM. Disk products provide only a limited area of tissue material coverage.
Another commercially available device sandwiches the microscope slide between larger rigid plates and has a silicone pad over the slide with holes in the pad corresponding to the areas where the tissue is affixed. Holes with removable plugs in the top plate allow test solutions to be added to the chambers formed by the pad. This product is called the Thermo-Slide.TM. Block, available from Elmeco Engineering, Rockville, Md. The Thermo-Slide.TM. Block product is designed to passively rest on the surface of a separate thermal plate or thermal cycler. The Thermo-Slide.TM. Block product has a number of limitations. It holds only two slides and has a thermal mass sufficient to render thermal cycling quite slow and, therefore, provide poorly defined thermal profiles on the slides.
A factor common to the above methods is the requirement of the user to manipulate each slide in one or more operations in order to control evaporation. Any method and apparatus which eliminates user handling of the slides would be beneficial not only for a particular procedure but also in the future development of automated slide handling systems.
In summary, therefore, the current methods and apparatus used to control evaporation during minimal volume histological procedures on microscope slides suffer from a variety of deficiencies. These include: a) limited thermal range; b) inability to use for thermal cycling procedures; c) tedious application; d) messy to use and clean-up afterwards; e) possible adverse affects on the test procedure; f) a requirement for special microscope slides; g) limited tissue material coverage; h) a requirement for expensive equipment and/or disposables; and, I) resistance to automated handling. Therefore, what is desired in method and apparatus to control evaporation which eliminates or substantially reduces many of the above deficiencies, and yet remains a cost-effective alterative to the prior art methods and apparatus discussed above.